Transgenic plants expressing puroindolines and methods for...

Multicellular living organisms and unmodified parts thereof and – Method of introducing a polynucleotide molecule into or... – The polynucleotide confers pathogen or pest resistance

Reexamination Certificate

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C800S301000, C800S320000, C800S320100, C800S320200, C800S320300, C800S317400, C435S419000, C435S411000, C435S412000

Reexamination Certificate

active

06600090

ABSTRACT:

FIELD OF THE INVENTION
This invention relates to pest control in plants. More specifically, this invention relates to methods for producing transgenic plant cells, plant tissues and whole plants which express effective anti-pathogen proteins and the transgenic cells, tissues and plants produced by such methods. Even more specifically, this invention relates to methods for producing transgenic plant cells, plant tissues and whole plants which express puroindoline proteins and the transgenic cells, tissues and plants produced by such methods. This invention also relates to isolated nucleic acid molecules which include a promoter operably linked to nucleic acids coding for one or more puroindoline proteins.
BACKGROUND
All publications and patent applications herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.
Higher plants primarily use three methods to limit or decrease the growth of pathogens: 1) the hypersensitive or gene-for-gene response; 2) inducible responses such as systemic acquired resistance; and 3) via antimicrobial peptides.
Anti-microbial peptides act as a type of innate immunity which limits pathogen growth and spread. This mechanism may play an important role in a plant's natural defenses against pathogens. Thionins, defensins, and non-specific lipid transfer proteins (nsLTPs) are the most common plant proteins reported to have anti-microbial properties (reviewed in Garcia-Olmedo et al., 1995; Broekaert et al., 1997).
Most experiments utilizing plants transgenic for anti-microbial peptides have assessed the effects of the over-expression of endogenous or heterologous anti-microbial proteins. For example, transgenic tobacco plants with constitutive expression of the barley LTP2 protein (Molina and Garcia-Olmedo, 1997) or the barley alpha-thionin (Carmona et al., 1993) showed much reduced bacterial pathogen infection on leaf tissue. Over-expression of endogenous thionin genes in
Arabidopsis thaliana
(Epple et al., 1997) also resulted in reduced wilt symptoms upon infection by
Fusarium oxysporum
. Molina et al. (1997
, Plant J.
12(3):669-675) report the expression of the barley lipid transfer protein LTP2 in transgenic tobacco. Some of these genes have been demonstrated to be inducible by pathogen infection (Molina et al., 1996).
Synergistic enhancement of anti-microbial properties has been demonstrated in vitro for a number of different anti-microbial proteins (Dubreil et al., 1998; Terras et al., 1993). The effects of multiple anti-microbial proteins may be expected to be at least additive.
There is a continuing need for alternative and supplementary methods of protecting plants from plant pests, including plant pathogens.
This invention provides for the control of pests via introduction of the puroindoline a gene (pinA) and the puroindoline b gene (pinB) into plant cells, plant tissues and plants. The constitutive protection offered by the PINA and PINB proteins, either singly or together, is surprising since these proteins are not part of an inducible defense found naturally in plants. Assessment of the inhibitory effect of the PINA and PINB proteins on pathogen growth on leaf tissue as demonstrated in the present invention is also unexpected since pinA and pinB are not expressed at all in normal leaf tissue (i.e., no puroindoline gene homologues are expressed in leaf tissue.). Prior to the present invention, no demonstration of in vivo anti-microbial properties has been demonstrated for the puroindolines. Thus, the present invention provides a new and important set of tools and methods for the protection of plants to pests which affect plant growth and yield.
SUMMARY OF THE INVENTION
This invention provides plant cells, plant tissues and plants transgenic for nucleic acids encoding puroindolines.
This invention further provides plant cells, plant tissues and plants transgenic for nucleic acids which hybridize under high stringency conditions with nucleic acids encoding puroindolines.
This invention also provides plant cells, plant tissues and plants transgenic for nucleic acids encoding fragments of puroindolines wherein the fragments retain at least one biological activity of the puroindolines.
This invention further provides plant cells, plant tissues or plants transgenic for recombinant DNA sequences encoding either or both of puroindolineA and puroindolineB.
This invention also provides plants which are transgenic for any of the above-listed nucleic acids, wherein the transgenic plants are capable of exhibiting a reduction in plant damage of greater than about 5% when compared to the corresponding non-transgenic plants following exposure of the transgenic plants and the corresponding non-transgenic plants to pests capable of damaging the plants. More specifically, this invention provides such transgenic plants wherein the transgenic plants are capable of exhibiting a reduction in plant damage of between about 5% and about 10%, or of between about 10% and about 20%, or of between about 20% and about 30%, or of between about 30% and about 40%, or of between about 40% and about 50%, or of between about 50% and about 60%, or of between about 60% and about 70%, or of between about 70% and about 80%, or of between about 80% and about 90%, or of between about 90% and about 100%, when compared to the corresponding non-transgenic plants following exposure of the transgenic plants and the corresponding non-transgenic plants to pests capable of damaging the plants. The pests which may be effectively controlled using the transgenic plants of the present invention include, but are not limited to, fungi, bacteria, viruses, nematodes, insects and mites. Of the insect and mite pests, the ones that are of particular importance to the present invention, include, but are not limited to, thoses that carry plant pathogens.
This invention provides monocotyledonous and dicotyledonous plants comprising either or both of puroindolineA and puroindolineB present in leaf tissue in an amount effective to reduce disease symptoms in response to infection by a pathogen. Such plants include, but are not limited to, maize, rice, wheat, barley, rye, canola, potatoes, tomatoes, sweet potatoes, sugar beets, tobacco, and cotton. More specifically this invention provides such plants wherein the pathogen is a fungus.
This invention further provides isolated nucleic acid molecules comprising nucleic acids operatively linked to constitutive or inducible promoters in a manner effective for expression of the nucleic acids, wherein the nucleic acids are selected from the group consisting of nucleic acids encoding one or more puroindolines, nucleic acids which hybridizes under high stringency conditions to the nucleic acids encoding puroindolines, and nucleic acids encoding fragments of a puroindoline wherein the fragments retain at least one biological activity of a puroindoline. This invention also provides methods of producing transformed plant cells, plant tissues or plants by transforming the plant cells, plant tissues or plants with such isolated nucleic acid molecules. This invention further provides methods of crossing the transformed plants to different plants, harvesting the resultant seeds, and planting and growing the harvested seeds.
DETAILED DESCRIPTION OF THE INVENTION
I. Definitions
As used herein, the term “allele” means any of several alternative forms of a gene.
As used herein, the term “crop plant” means any plant grown for any commercial purpose, including, but not limited to the following purposes: seed production, hay production, ornamental use, fruit production, berry production, vegetable production, oil production, protein production, forage production, silage, animal grazing, golf courses, lawns, flower production, landscaping, erosion control, green manure, improving soil tilth/health, producing pharmaceutical products/drugs, producing food additives, smoking products, pulp production and wood production. Particular crop plants of

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